The present invention concerns a procedure for extracting endoproteolytic hatching enzymes (zonases) in waste water from hatcheries producing Atlantic salmon larvae, and in addition, it establishes a simple procedure for obtaining up to sequence-grade purity of these special endoproteases, which turn out to possess rather unique proteolytic characteristics.
Proteases in purified states are increasingly used in research, in laboratory and clinical analysis, and in food production procedures. Demand is increasing, especially for enzymes with properties commensurate with specific applications. This has stimulated quests for new sources of proteases which allow safe, sustainable and economical modes of production.
Here we exploit a new, rich source of endoproteases connected to aquaculture of Atlantic salmon. An essential aspect of this industry is the hatchery-production of developing eggs, which hatch to yield larvae. Hatching is accomplished by embryos producing endoproteases, which, when secreted, effectively and specifically split the eggshell open to allow the larva to swim out and start life on its own (Refs.: Yamagami 1988; Walther 1993).
The critical enzymes behind fish hatching have only been characterized in a few fishes, and in almost all cases, these zonases have been interpreted to be metallo-proteases (Refs.: Hagenmaier 1974; Ohzu and Kasuya 1979; Schoots and Denucxc3xa9 1981; DiMichele et al. 1981; Yasumasu et al. 1989a, b; Araki and Onozato 1990; Hung et al. 1997). Gene structure of some zonase-type enzymes has only recently become available (Ref.: Yasumasu et al 1992).
Putative hatching enzymes have also been reported in invertebrates, where again most such enzymes have been interpreted as being metalloproteases (e.g. Barrett and Edward 1976; Lepage and Gache 1989; Roe and Lennarz 1990). However, a few strong cases for serine protease-like zonases have been reported (e.g. Post et al. 1988). Conversely, among higher vertebrates than fish, putative hatching enzymes have also been reported. For instance, both Urch and Hedrick (1981; concerning amphibians) and Yamazaki et al (1994; concerning mouse) reported zonases which appeared to be serine proteases. The biological and biochemical rationale behind two different types of zonases among hatching animals is at present not fully understood.
In Atlantic salmon, we have found the predominant zonases to be serine proteases. These are present in large amounts in the waste waters and hatching fluids of salmon eggs. In this crude aqueous state, salmon zonases may be effectively readied for purification by conventional techniques. This source of zonases offers a great advantage compared to isolation of enzymes from whole embryos, since it effectively obviates complications from extraneous biomaterial (eggshells, embryos and larvae). Thus, enzyme purification becomes greatly simplified.
An additional advantage is that the developmentally-staged salmon eggs may be transferred to minimal volumes of water prior to hatching. When highly synchronous hatching is induced by elevated (room) temperatures, or by deoxygenaton (Oppen-Berntsen et al. 1990), this yields a small volume of highly concentrated preparation of crude zonases.
A further essential aspect of this procedure is that, despite the increasing concentration of the proteolytic zonases, the stability of its resident zonases was observed to remain intact. Furthermore, it is important to note that this procedure yields zonase enzymes in a medium of almost pure water, containing at most 1 mM NaCl, but where zonases nevertheless possess and retain full enzymatic integrity over time. This preparation is therefore a valuable starting material for subsequent preparations of proteolytic zonases in various degree of purification, up to sequence-grade purity.